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. 2019 May 28;10:2350. doi: 10.1038/s41467-019-10359-x

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — SLIT2 induces ENDOA2-dependent VEGFR2 internalization. a Cell surface biotinylation assay of VEGFR2 internalization in response to SLIT2 (6 nM) in Control (Ctrl), ROBO1/2 and ENDOA2 siRNA silenced HUVECs. VEGFR2, ROBO1, ENDOA2, and ACTIN expression from the total cell lysate are shown as loading controls (input). Surf: surface expression. b Quantification of internalized VEGFR2 normalized to VEGFR2 surface expression before stimulation (N = 7 independent experiments; two-way ANOVA test: *P < 0.05, **P < 0.01, ***P < 0.001, ns P > 0.05). c, d Antibody feeding assay to measure VEGFR2 internalization in response to SLIT2 (6 nM) in Ctrl, ROBO1/2, and ENDOA2 siRNA silenced HUVECs (c) and mLECs from EndoA2^+/+ and EndoA2^−/− mice (d). e, f Quantification of internalized VEGFR2 fluorescent intensity shown in c and d, respectively (N = 4 independent experiment, at least 10³ cells analyzed per experiment; e one-way ANOVA and f Mann–Whitney U test: *P < 0.05). g SIM images of HUVEC lamellipodia stained for ENDOA2 before and after SLIT2 stimulation (6 nM for 2′30″). h SLIT2 effects on EPA formation at the cell migration front (N = 16–17 cells per group analyzed from three independent experiments; t-test: ***P < 0.001). i SLIT2 increases the pixel overlap between VEGFR2 and ENDOA2 fluorescent signals (N = 8–10 cells per group analyzed from three independent experiments; Mann–Whitney U test: ***P < 0.001). j SIM images of HUVEC lamellipodia stained for ENDOA2, VEGFR2, and CLATHRIN heavy chain after SLIT2 stimulation (6 nM for 2′30″). k–m Overlapping pixels between VEGFR2/ENDOA2 (k), VEGFR2/CLATHRIN (l), and ENDOA2/CLATHRIN (m) from the image presented in j are shown in white. Boxed areas are magnified to highlight VEGFR2/ENDOA2, VEGFR2/CLATHRIN, or ENDOA2/CLATHRIN overlap (white). n Quantification of overlap between VEGFR2/ENDOA2 and VEGFR2/CLATHRIN fluorescent signals (N = 8–10 cells per group analyzed from three independent experiments; Mann–Whitney U test: **P < 0.01) (right panel). Error bars represent mean ± s.e.m. Scale bars: c and d 20μm, g 2μm, j 1μm